Mesenchymal stem cells (MSCs) are multilineage cells able to differentiate into other cell types. MSCs derived from bone marrow or compact bones are the most accessible stem cells used in tissue engineering. Therefore, the aim of this study was to isolate, characterize and cryopreserve MSCs of endangered Oravka chicken breed. MSCs were obtained from compact bones of the femur and tibiotarsus. MSCs were spindle-shaped and were able to differentiate into osteo-, adipo-, and chondrocytes under the specific differentiation conditions. Furthermore, MSCs were positive for surface markers such as CD29, CD44, CD73, CD90, CD105, CD146 and negative for CD34CD45 by flow cytometry. Moreover, MSCs demonstrated high positivity of "stemness" markers aldehyde dehydrogenase, alkaline phosphatase as well as for intracellular markers vimentin, desmin, α-SMA. Subsequently, MSCs were cryopreserved using 10% dimethyl sulfoxide in liquid nitrogen. Based on the results from the viability, phenotype, and ultrastructure assessment we can concluded that the MSCs were not negatively affected by the cryopreservation. Finally, MSCs of endangered Oravka chicken breed were successfully stored in animal gene bank, thus making them a valuable genetic resource.

Department of Zoology and Anthropology Faculty of Natural Sciences Constantine the Philosopher University in Nitra Nitra Slovakia

Institute of Farm Animal Genetics and Reproduction NPPC Research Institute for Animal Production in Nitra Nitra Slovakia

Institute of Farm Animal Genetics and Reproduction NPPC Research Institute for Animal Production in Nitra Nitra Slovakia; Department of Botany and Genetics Faculty of Natural Sciences Constantine the Philosopher University in Nitra Nitra Slovakia

Institute of Farm Animal Genetics and Reproduction NPPC Research Institute for Animal Production in Nitra Nitra Slovakia; Institute of Biotechnology Faculty of Biotechnology and Food Science Slovak University of Agriculture in Nitra Nitra Slovakia

Institute of Farm Animal Genetics and Reproduction NPPC Research Institute for Animal Production in Nitra Nitra Slovakia; Laboratory of Animal Immunology and Biotechnology Department of Animal Morphology Physiology and Genetics Faculty of AgriSciences Mendel University in Brno Brno Czech Republic

Laboratory of Animal Immunology and Biotechnology Department of Animal Morphology Physiology and Genetics Faculty of AgriSciences Mendel University in Brno Brno Czech Republic

Laboratory of Animal Physiology Department of Animal Morphology Physiology and Genetics Faculty of AgriSciences Mendel University in Brno Brno Czech Republic

Zobrazit více v PubMed

Adhikari R., Chen C., Waters E., West F.D., Kim W.K. Isolation and differentiation of mesenchymal stem cells from broiler chicken compact bones. Front. Physiol. 2019;9:1892. PubMed PMC

Almalki S.G., Agrawal D.K. Key transcription factors in the differentiation of mesenchymal stem cells. Differentiation. 2016;92:41–51. PubMed PMC

Arrizabalaga J.H., Nollert M.U. Properties of porcine adipose-derived stem cells and their applications in preclinical models. Adipocyte. 2017;6:217–223. PubMed PMC

Aviagen . Aviagen Inc.; Huntsville, AL: 2014. AviagenRoss 308: Broiler Management Handbook.

Ayala-Cuellar A.P., Kang J.H., Jeung E.B., Choi K.C. Roles of mesenchymal stem cells in tissue regeneration and immunomodulation. Biomol. Ther. 2019;27:25–33. PubMed PMC

Bai C., Hou L., Ma Y., Chen L., Zhang M., Guan W. Isolation and characterization of mesenchymal stem cells from chicken bone marrow. Cell Tissue Bank. 2013;14:437–451. PubMed

Bárcia R.N., Santos J.M., Teixeira M., Filipe M., Pereira A.R.S., Ministro A., Cruz H. Umbilical cord tissue-derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing. Cytotherapy. 2017;19:360–370. PubMed

Bharti M.K., Bhat I.A., Pandey S., Shabir U., Peer B.A., Indu B., Sharma G.T. Effect of cryopreservation on therapeutic potential of canine bone marrow derived mesenchymal stem cells augmented mesh scaffold for wound healing in guinea pig. Biomed. Pharmacother. 2020;121 PubMed

Blackburn H.D. The National Animal Germplasm Program: challenges and opportunities for poultry genetic resources. Poult. Sci. 2006;85:210–215. PubMed

Bourebaba L., Michalak I., Baouche M., Kucharczyk K., Marycz K. Cladophora glomerata methanolic extract promotes chondrogenic gene expression and cartilage phenotype differentiation in equine adipose-derived mesenchymal stromal stem cells affected by metabolic syndrome. Stem. Cell Res. Ther. 2019;10:1–20. PubMed PMC

Cruz F.F., Rocco P.R.M. The potential of mesenchymal stem cell therapy for chronic lung disease. Expert. Rev. Respir. Med. 2020;14:31–39. PubMed

Dominici M.L.B.K., Le Blanc K., Mueller I., Slaper-Cortenbach I., Marini F.C., Krause D.S., Horwitz E.M. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–317. PubMed

Elashry M.I., Gegnaw S.T., Klymiuk M.C., Wenisch S., Arnhold S. Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for equine adipose tissue-derived mesenchymal stem cells. Acta Histochem. 2019;121:344–353. PubMed

Gandini G.C., Oldenbroek J.K. Genebanks and the Conservation of Farm Animal Genetic Resources. DLO-Institute for Animal Science and Health (ID-DLO), Instituut voor Veehouderij en Diergezondheid, Research Branch Zeist;  Lelystad, Netherlands: 1999. Choosing the conservation strategy; pp. 11–31.

Gayathri V., Harikrishnan V., Mohanan P.V. Integration of rabbit adipose derived mesenchymal stem cells to hydroxyapatite burr hole button device for bone interface regeneration. Int. J. Biomater. 2016;2016:1–9. PubMed PMC

Gil A., Plaza-Diaz J., Mesa M.D. Vitamin D: classic and novel actions. Ann. Nutr. Metab. 2018;72:87–95. PubMed

Golchin A., Farahany T.Z., Khojasteh A., Soleimanifar F., Ardeshirylajimi A. The clinical trials of mesenchymal stem cell therapy in skin diseases: an update and concise review. Curr. Stem Cell Res. Ther. 2018;14:22–33. PubMed

Kawai T., Katagiri W., Osugi M., Sugimura Y., Hibi H., Ueda M. Secretomes from bone marrow-derived mesenchymal stromal cells enhance periodontal tissue regeneration. Cytotherapy. 2015;17:369–381. PubMed

Khan I.U., Yoon Y., Choi K.U., Jo K.R., Kim N., Lee E., Kweon O.K. Therapeutic effects of intravenous injection of fresh and frozen thawed ho-1-overexpressed ad-mscs in dogs with acute spinal cord injury. Stem Cells Int. 2019;2019 PubMed PMC

Khatri M., O'Brien T.D., Sharma J.M. Isolation and differentiation of chicken mesenchymal stem cells from bone marrow. Stem Cells Dev. 2009;18:1485–1492. PubMed

Kim H.K., Lee S.G., Lee S.W., Oh B.J., Kim J.H., Kim J.A., Joe Y.A. A subset of paracrine factors as efficient biomarkers for predicting vascular regenerative efficacy of mesenchymal stromal/stem cells. Stem Cells. 2019;37:77–88. PubMed

Kulikova B., Kovac M., Bauer M., Tomkova M., Olexikova L., Vasicek J., Chrenek P. Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification. Acta Histochem. 2019;121:491–499. PubMed

Li D., Chen Z., Chen S., Ji H., Zhan X., Luo D., Luo H., Wang B. Chicken mesenchymal stem cells as feeder cells facilitate the cultivation of primordial germ cells from circulating blood and gonadal ridge. Stem Cell Discov. 2019;9:1–14.

Lin C.S., Xin Z.C., Dai J., Lue T.F. Commonly used mesenchymal stem cell markers and tracking labels: limitations and challenges. Histol. Histopathol. 2013;28:1109. PubMed PMC

Lohan P., Murphy N., Treacy O., Lynch K., Morcos M., Chen B., Ritter T. Third-party allogeneic mesenchymal stromal cells prevent rejection in a pre-sensitized high-risk model of corneal transplantation. Front. Immunol. 2018;9:2666. PubMed PMC

Machado A.K., Homrich S.G., Rodrigues C.C.R., Azzolin V.F., Duarte M.M.M.F., Pillar D.M., Da Cruz I.B.M. Human adipose-derived stem cells obtained from lipoaspirates are highly susceptible to hydrogen peroxide mediated cytogenotoxicity. Arch. Biosci. Health. 2019;1:11–28.

Majumdar M.K., Thiede M.A., Mosca J.D., Moorman M., Gerson S.L. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J. Cell. Physiol. 1998;176:57–66. PubMed

Mendelsohn R. The challenge of conserving indigenous domesticated animals. Ecol. Econ. 2003;45:501–510.

Muir W.M., Wong G.K.S., Zhang Y., Wang J., Groenen M.A., Crooijmans R.P., Cheng H.H. Genome-wide assessment of worldwide chicken SNP genetic diversity indicates significant absence of rare alleles in commercial breeds. Proc. Natl. Acad. Sci. 2008;105:17312–17317. PubMed PMC

Nakamura Y., Ishikawa H., Kawai K., Tabata Y., Suzuki S. Enhanced wound healing by topical administration of mesenchymal stem cells transfected with stromal cell-derived factor-1. Biomaterials. 2013;34:9393–9400. PubMed

Oh E.J., Lee H.W., Kalimuthu S., Kim T.J., Kim H.M., Baek S.H., Ahn B.C. In vivo migration of mesenchymal stem cells to burn injury sites and their therapeutic effects in a living mouse model. J. Control. Release. 2018;279:79–88. PubMed

Palumbo P., Lombardi F., Siragusa G., Cifone M.G., Cinque B., Giuliani M. Methods of isolation, characterization and expansion of human adipose-derived stem cells (ASCs): an overview. Int. J. Mol. Sci. 2018;19:1897. PubMed PMC

Rogulska O., Tykhvynska O., Revenko O., Grischuk V., Mazur S., Volkova N., Petrenko Y. Novel cryopreservation approach providing off-the-shelf availability of human multipotent mesenchymal stromal cells for clinical applications. Stem Cells Int. 2019;2019 PubMed PMC

Sacchetti B., Funari A., Michienzi S., Di Cesare S., Piersanti S., Saggio I., Bianco P. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell. 2007;131:324–336. PubMed

Salmenkari H., Laitinen A., Forsgård R.A., Holappa M., Linden J., Pasanen L., Nystedt J. The use of unlicensed bone marrow-derived platelet lysate-expanded mesenchymal stromal cells in colitis: a pre-clinical study. Cytotherapy. 2019;21:175–188. PubMed

Selvasandran K., Makhoul G., Jaiswal P.K., Jurakhan R., Li L., Ridwan K., Cecere R. A tumor necrosis factor-α and hypoxia-induced secretome therapy for myocardial repair. Ann. Thorac. Surg. 2018;105:715–723. PubMed

Sidney L.E., Branch M.J., Dunphy S.E., Dua H.S., Hopkinson A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells. 2014;32:1380–1389. PubMed PMC

Somal A., Bhat I.A., Singh A.P., Panda B.S., Desingu P.A., Pandey S., Sharma G.T. Impact of cryopreservation on caprine fetal adnexa derived stem cells and its evaluation for growth kinetics, phenotypic characterization, and wound healing potential in xenogenic rat model. J. Cell. Physiol. 2017;232:2186–2200. PubMed

Teresa Conconi M., Di Liddo R., Tommasini M., Calore C., Paolo Parnigotto P. Phenotype and differentiation potential of stromal populations obtained from various zones of human umbilical cord: an overview. Open Tissue Eng. Regen. Med. J. 2011;4: 6– 20.

Tirpáková M., Vašíček J., Svoradová A., Baláži A., Tomka M., Bauer M., Chrenek P. Phenotypical characterization and neurogenic differentiation of rabbit adipose tissue-derived mesenchymal stem cells. Genes. 2021;12:431. PubMed PMC

Tixier-Boichard M., Bordas A., Rognon X. Characterisation and monitoring of poultry genetic resources. Worlds Poult. Sci. J. 2009;65:272–285.

Vašíček J., Kováč M., Baláži A., Kulíková B., Tomková M., Olexiková L., Chrenek P. Combined approach for characterization and quality assessment of rabbit bone marrow-derived mesenchymal stem cells intended for gene banking. New Biotechnol. 2020;54:1–12. PubMed

Vassalli G. Aldehyde dehydrogenases: not just markers, but functional regulators of stem cells. Stem Cells Int. 2019;2019:1–15. PubMed PMC

Weigend S., Romanov M.N. Current strategies for the assessment and evaluation of genetic diversity in chicken resources. Worlds Poult. Sci. J. 2001;57:275–288.

Weigend S., Romanov M.N. The world watch list for domestic animal diversity in the context of conservation and utilisation of poultry biodiversity. Worlds Poult. Sci. J. 2002;58:411–425.

Xu T., Lv Z., Chen Q., Guo M., Wang X., Huang F. Vascular endothelial growth factor over-expressed mesenchymal stem cells-conditioned media ameliorate palmitate-induced diabetic endothelial dysfunction through PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathway. Biomed. Pharmacother. 2018;106:491–498. PubMed

Ye J., Coulouris G., Zaretskaya I., Cutcutache I., Rozen S., Madden T.L. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 2012;13:1–11. PubMed PMC

Yea J.H., Park J.K., Kim I.J., Sym G., Bae T.S., Jo C.H. Regeneration of a full-thickness defect of rotator cuff tendon with freshly thawed umbilical cord-derived mesenchymal stem cells in a rat model. Stem Cell Res. Ther. 2020;11:1–13. PubMed PMC

Zhan X.S., El-Ashram S., Luo D.Z., Luo H.N., Wang B.Y., Chen S.F., Ji H.Q. A comparative study of biological characteristics and transcriptome profiles of mesenchymal stem cells from different canine tissues. Int. J. Mol. Sci. 2019;20:1485. PubMed PMC

Zomer H.D., Roballo K.C., Lessa T.B., Bressan F.F., Gonçalves N.N., Meirelles F.V., Ambrósio C.E. Distinct features of rabbit and human adipose-derived mesenchymal stem cells: implications for biotechnology and translational research. Stem Cells Cloning: Adv. Appl. 2018;11:43–54. PubMed PMC